Etanercept is a biological inflammation modulator that functions as a competitive inhibitor of TNF-α, binding to cell surface TNF-α receptor, to inhibit TNF-α mediated immune responses. Etanercept is a macromolecule with a molecular weight of approximately 150 kDa, and is a homodimer of two Fc fusion proteins linked by a disulfide bond, each Fc fusion protein consisting of a human soluble p75 TNF receptor coupled to the Fc portion of human immunoglobulin G subclass 1 (Goldenberg, Clinical Therapeutics, 21(1): 75-87, 1999; Moreland et al., Ann. Intern. Med., 130(6): 478-486, 1999).
This prototypic fusion protein was first synthesized in the early 1990s by Bruce A. Beutler at the University of Texas Southwestern Medical Center, and marketed by Amgen under the trade name of Enbrel in 2002. Etanercept is a TNF-α inhibitor used to treat rheumatoid arthritis, psoriasis, and ankylosing spondylitis, and under clinical trials for the treatment of vasculitis, Alzheimer's disease, and Crohn's disease.
Like protein drugs, antibody drugs have a very short half life, and chemical and physical denaturation can be easily caused by unfavorable temperature, shear stress, vibration, freeze-thawing, UV exposure, excessive pH change, organic solvents, and microbial contamination. Chemical denaturation includes dimer dissociation, oxidation, deamidation, isomerization, and polymerization, which are influenced by the amino acids constituting the antibody and conditions of the solvent containing the antibody (salt, pH and temperature). Physical denaturation includes loss of tertiary structure, covalent/non-covalent aggregation and adhesion of monomers, which are influenced by hydrophobic patches on the protein surface changed by antibody-containing surrounding environments such as solvents, complex protein structures such as charge distribution, and thermal stability. The physical or chemical denaturation of an antibody causes loss of its physiological activities. Since the denaturation is an irreversible process, antibodies, once denatured, may not recover their native properties to the initial state, leading to a reduction in their therapeutic efficacies. It has been also suggested that aggregation of monomers causes immune responses. Therefore, many studies have been conducted on antibody formulations containing a physiologically effective amount without aggregates (Ishikawa et al., Biol. Pharm. Bull., 33(8): 1413-1417, 2010; Levin et al., The Development of Therapeutic Monoclonal Antibody Products, editors. Boston (Mass.): BioProcess Technology Consultants, Inc, Chapter 9).
There are many methods available for preventing protein denaturation in liquid formulations. In some protein drugs, the stability problems are addressed via lyophilization. For instance, U.S. Pat. No. 7,592,004 discloses a lyophilized formulation of daclizumab that is stabilized using 5 to 25 mM of a histidine buffer solution (pH 5.5 to 6.5), 0.005 to 0.03% of a non-ionic surfactant, polysorbate and 100 to 300 mM of a non-reducing sugar, sucrose. U.S. Patent Publication No. 2010-0158925 discloses a lyophilized formulation of cetuximab that is stabilized using a histidine buffer solution and lactobionic acid. However, the lyophilization process generates freezing and drying stresses such as formation of ice crystals, pH change, and high concentration of solute, and these stresses may cause antibody denaturation. In addition, since a large-capacity lyophilizer is needed for the lyophilization process during the production, high production costs arise during a large scale of production. Dissolving the lyophilized product in sterile aqueous media for reconstitution before use also poses an inconvenience.
As an alternative to solve these limitations, a stabilizer is added in liquid formulations for the improvement of antibody stability. Surfactants, serum albumins, polysaccharides, amino acids, polymers, salts or the like are known as stabilizers for proteins including antibodies (Wang, Int. J. Pharm., 185: 129-188, 1999; Wang et al., J. Pharm. Sci., 96(1): 1-26, 2007).
US Patent Publication No. 2011-0070231 discloses a stable liquid composition of IgG antibody, in which the stable pharmaceutical liquid formulation includes 50 mg/mL or more of daclizumab in 20 to 60 mM of a succinate buffet solution (pH 5.5 to 6.5), 0.02 to 0.04% of polysorbate, and 75 to 150 mM of sodium chloride.
US Patent Publication No. 2011-0020328 discloses a therapeutically effective amount of anti-CD20 antibody formulation, in which the pharmaceutically stable composition includes 10 to 100 mM of sodium acetate, 25 to 100 mM of sodium chloride, 0.5 to 5% of arginine free base, 0.02 to 0.2 mM of EDTA and 0.01 to 0.2% of polysorbate 80, and its pH is 5.0 to 7.0.
U.S. Pat. No. 7,785,592 discloses a stable formulation, in which 75 mg/mL or more of palivizumab is stabilized by using a histidine buffer solution and glycine without ionic salts and a surfactant, and its stability is maintained at 2 to 8° C. for at least 15 months.
U.S. Pat. No. 6,991,790 discloses a stable aqueous pharmaceutical formulation, including a therapeutically effective amount of an antibody, an acetate buffer solution of approximately pH 4.8 to 5.5, a surfactant, and a polyol without an isotonic agent such as sodium chloride.
U.S. Pat. No. 7,648,702 discloses a liquid formulation, in which a fusion protein of the human p75 tumor necrosis factor receptor linked to the Fc portion of the human immunoglobulin G1 (IgG1) is stabilized by using approximately 10 to 200 mM of L-arginine as an aggregation preventing agent. This patent is the only technique of using etanercept as an active ingredient.
In order to prepare stable formulations, however, appropriate stabilizers should be used considering the physicochemical properties of each active ingredient. When the stabilizers are used in combination, competition therebetween and adverse effects may lead to undesirable effects. In addition, the concentrations of antibodies should be within the range suitable for the stabilization, and their concentrations are relatively higher than those of protein drugs. Thus, much effort and caution are required to stabilize antibodies in solutions (Shire et al., J. Pharm. Sci., 93(6): 1390-1402, 2004).
There are few studies on the stable liquid formulations of etanercept, and the only method for stabilizing etanercept that the inventors are aware of is to use L-arginine in the liquid formulation. Therefore, there is an urgent need to develop a new liquid formulation which is able to stably maintain the activity of etanercept for a long period and which is more effective in etancercept stabilization than the known formulation comprising L-arginine.